1. Field of the Invention
The present invention is generally directed to voltage-gated sodium channel NaV1.8 from Pan troglodytes (chimp). The invention further describes methods and compositions for the stable expression of chimp NaV1.8 sodium channels and methods of use of such compositions for identifying compounds that modulate the activity of sodium channels.
2. Background of the Related Art
The electrical activity of neuronal and muscle cells are governed by the activity of sodium channels on the plasma membrane of such cells. Rapid entry of sodium ions into the cell through such a channel causes depolarization of the membrane and generation of an action potential. Entry of sodium ions through sodium channels in response to a voltage change on the plasma membrane in excitable cells plays a functional role in the excitation of neurons in the central nervous system and the peripheral nervous system.
Sodium channels are voltage-gated transmembrane proteins that form ion channels within the membrane and have been the target of significant pharmacologic study, due to their potential role in a variety of pathological conditions. These sodium channels are responsible for the cellular uptake of sodium during the transmission of an electrical signal in cell membranes. The channels are members of a multigene family of proteins and are typically composed of a number of subunits. Typically, the pore of the channel is formed by the α-subunit and there are four accessory β-subunits, termed β1, β2, β3 and β3.
The β-subunits are involved in the modulation of the activity of sodium channel but the α-subunit is all that is required for the channel to form a functional ion pore. Co-expression of the β-subunits with the α-subunit has been shown to produce a more positive membrane potential. Further not all of the β-subunits are required, for example, it has been shown that the β3-subunits alone is sufficient to cause an increase in sodium current (Qu et al., Mol. Cell. Neurosci., 18(5):570-80 (2001)).
The amino acid sequence of the sodium channel has been evolutionarily conserved. The channel is comprised of a signal polypeptide containing four internal repeats (domains I-IV). Each domain folds into six transmembrane α-helices or segments, of which five are hydrophobic and one is a highly-charged domain containing lysine and arginine residues (S4 segment). The highly-charged S4 segment is involved in the voltage gating properties of the sodium channel. The positively-charged side chains of the amino acids of the S4 segment are thought to be paired with the negatively-charged side chains of the other five segments such that upon membrane depolarization there is a shift in the position of one of the helices relative to the other resulting in an opening of the channel.
There are numerous variants of sodium channel α-subunit. These variants may be classified according to their sensitivity to tetrodotoxin (TTX). Those subunits that are inhibited by nanomolar quantities of TTX are classified as tetrodotoxin-sensitive channels, whereas those that require at least micromolar quantities of TTX for inhibition are referred to as tetrodotoxin-insensitive (1-5 micromolar). Those channels that require greater that 100 micromolar quantities of the TTX are termed tetrodotoxin-resistant. TTX is a toxin that blocks the conduction of nerve impulses along the axons and leads to paralysis. It binds to sodium channels and blocks the flow of sodium ions. It is believed that the positively-charged group of the toxin interacts with a negatively-charged carboxylate at the mouth of the channel on the extracellular side of the membrane thereby blocking the conductance of the pathway.
It has been noted that following nerve injury there is hyperexcitability (or an increased rate of spontaneous impulse firing in neurons) in peripheral sensory ganglia. It has been suggested that this hyperexcitability in neurons is due to altered sodium channel expression in some chronic pain syndromes (Tanaka et al., Neuroreport; 9 (6): 967-72 (1998)). Increased numbers of sodium channels leading to inappropriate, repetitive firing of the neurons have been reported in the tips of injured axons in various peripheral nervous tissues such as the DRG, which relay signals from the peripheral receptors to the central nervous system. Indeed, it has been noted that there is an increase in expression of an α1 Nav 1.3 subunit in axotomized DRG neurons together with elevated levels of α1 Nav1.1 and α1 Nav1.2 mRNAs (Waxman et al, Brain Res Mol Brain Res; 22 (1-4): 275-89 (1994)).
The peripheral input that drives pain perception is thought to depend upon the presence of functional voltage-gated sodium channels in peripheral nerves. It has been noted that there is a positive correlation between increased sodium channel expression in peripheral nerves. Some studies have also shown increased expression in association with neuropathic pain. In particular, it has been recognized that acute, inflammatory, and neuropathic pain can all be attenuated or abolished by local treatment with sodium channel blockers such as lidocaine. Remarkably, two voltage-gated sodium channel genes (NaV1.8 and Nav1.9) are expressed selectively in damage-sensing peripheral neurons, while a third channel (Nav1.7) is found predominantly in sensory and sympathetic neurons. An embryonic channel (NaV1.3) is also upregulated in damaged peripheral nerves and associated with increased electrical excitability in neuropathic pain states. Using antisense and knock-out studies, it has been shown that these sodium channels play a specialized role in pain pathways, and pharmacological studies (Wood et al., J Neurobiol., 61(1):55-71 (2004)).
Most patients with traumatic spinal cord injury (SCI) report moderate to severe chronic pain that is refractory, or only partially responsive, to standard clinical interventions (Balazy, Clin J Pain 8: 102-110 (1992); Turner et al., Arch Phys Med Rehabil 82: 501-509 (2001)). Experimental contusion SCI in rodents can produce long-lasting central neuropathic pain (Hulsebosch et al., J Neurotrauma 17: 1205-1217 (2000); Lindsey et al., Neurorehabil Neural Repair 14: 287-300 (2000); Hains et al., Neuroscience 116: 1097-1110 (2001); Mills et al., J Neurotrauma 18: 743-756 (2001)). In spinally injured animals, alterations in electrophysiologic properties of dorsal horn neurons (Hao et al., Pain 45: 175-185 (1991); Yezierski and Park, Neurosci Lett 157: 115-119 (1993); Drew et al., Brain Res 893: 59-69 (2001); Hains et al., Neuroscience 116: 1097-1110 (2003a); Hains et al., Brain Res 970: 238-241 (2003b)) are thought to contribute to changes in somatosensory responsiveness.
An analysis of human and chimp genomes reveals a difference of 1% between the two species. Mapping this critical difference could lead to important insights into functions of specific genes. NaV1.8 and NaV1.9 contain an amino acid sequence common to tetrodotoxin resistant Na+ channels and are localized in peripheral nociceptors. Recent patch-clamp experiments on dorsal root ganglion neurons from NaV1.8-knock-out mice unveiled an additional tetrodotoxin-resistant Na+ current. The demonstration of its dependence on NaV1.9 provides evidence for a specialized role of NaV1.9, together with NaV1.8, in pain sensation. (Ogata, N., and Ohishi, Y, Jpn J Pharmacol 88, 365-377 (2002)). Chronic axonal damage of sensory neurons often results in painful and dysaesthetic sensations. These positive sensory symptoms of peripheral nerve injury are produced by ectopic nerve impulses in the damaged neurons generated at the site of injury by sprouting axons (known as a neuroma) and also at the soma.
Sodium channels accumulate at the sites of sprouting after axonal damage (Devor, M., et al., J Neurosci 13, 1976-1992 (1993)) and pharmacological experiments reveal an important role for voltage-gated sodium channels in spontaneous electrogenesis in neuromas (Matzner, O., & Devor M., J Neurophysiol 72, 349-359 (1994)). A selective accumulation of NaV1.8 in injured nerve fibers in the rat (Novakovic et al., J Neurosci 18, 2174-2187 (1998)) and in damaged nerves and skin from patients with painful neuropathy (Coward et al., Pain 85, 41-50 (2000)) has been shown using subtype-specific antibodies. Further inhibiting the expression of NaV1.8 protein using antisense oligonucleotides reverses thermal and mechanical hyperalgesia produced by spinal nerve ligation in rats (Porreca et al., Proc Natl Acad Sci U S A 96, 7640-7644 (1999); Lai et al., Pain 95, 143-152 (2002)).
There are various sodium channels that remain to be characterized. Identification of such channels will facilitate further studies and identification and characterization of further isotype-specific antagonists of sodium channel blockers. Such sodium channel blockers or antagonists will be useful in the management of pain. Preferably, such analgesic agents are such that treatment of pain is facilitated without having deleterious side effects due to cardiac, central nervous system or neuromuscular complications.
The present invention is directed to a chimp sodium channel alpha subunit and methods and compositions for making and using the same. More specifically, one embodiment of the invention is directed to an isolated recombinant nucleic acid encoding a chimp sodium channel NaV1.8 polypeptide, wherein the polypeptide comprises a) a polypeptide sequence of SEQ ID NO: 2, b) a variant SEQ ID NO: 2 that is at least 90% identical to SEQ ID NO: 2 and has sodium channel activity; c) a NaV1.8 polypeptide that is encoded by a nucleic acid having the sequence of SEQ ID NO: 1, or d) a variant of SEQ ID NO: 2 encoded by a nucleic acid that hybridizes with a complementary strand of SEQ ID NO: 1 under stringent hybridization conditions wherein said variant has sodium channel activity. By “sodium channel activity” it is meant an activity associated with the uptake of sodium during the transmission of electrical signals in cell membranes.
The present invention is directed to a chimp sodium channel alpha subunit and methods and compositions for making and using the same. More specifically, one embodiment of the invention is directed to an isolated recombinant nucleic acid encoding a chimp sodium channel NaV1.8 polypeptide, wherein the polypeptide comprises a) a polypeptide sequence of SEQ ID NO: 2, b) a variant SEQ ID NO: 2 that is at least 90% identical to SEQ ID NO: 2 and has sodium channel activity; c) a NaV1.8 polypeptide that is encoded by a nucleic acid having the sequence of SEQ ID NO: 1, or d) a variant of SEQ ID NO: 2 encoded by a nucleic acid that hybridizes with a complementary strand of SEQ ID NO: 1 under stringent hybridization conditions wherein said variant has sodium channel activity. By “sodium channel activity” it is meant an activity associated with the uptake of sodium during the transmission of electrical signals in cell membranes.
Another embodiment of the invention describes an isolated recombinant nucleic acid encoding a chimp sodium channel NaV1.8 protein having the amino acid sequence of SEQ ID NO: 2. Also contemplated herein is an isolated recombinant nucleic acid comprising the sequence presented in SEQ ID NO: 1, the mature protein coding portion thereof, or a complement thereof. One preferred embodiment of the invention contemplates an isolated recombinant nucleic acid encoding a polypeptide of SEQ ID NO: 2. The nucleic acids described herein may be genomic DNA, cDNA, or RNA.
Another embodiment of the invention describes a compound 8 to 80 nucleotides in length targeted to a nucleic acid molecule encoding NaV1.8, wherein the compound specifically hybridizes with a nucleic acid molecule of SEQ ID NO: 1 and inhibits the expression of NaV1.8. In a related embodiment, the compound is 12 to 50, preferably 15 to 30, or more preferably 19 to 25 nucleotides in length.
In another embodiment, the compound is an antisense oligonucleotides, a DNA oligonucleotides, or an RNA oligonucleotide. In still another embodiment, at least a portion of the aforementioned compound hybridizes with RNA to form an oligonucleotide-RNA duplex. In yet another embodiment, the compound has at least 70%, at least 80%, at least 90%, or at least 95% complementarity with a nucleic acid molecule of SEQ ID NO 1 wherein said compound specifically hybridizes to and inhibits the expression of NaV1.8. In another embodiment, the compound has at least one modified internucleoside linkage, sugar moiety, or nucleotide.
Conservative variants of the sequences of the present invention are particularly contemplated, for example, the invention is directed to an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide that is a conservative variant of the amino acid sequences set forth in SEQ ID NO: 2, wherein the variant encodes a sodium channel α-subunit.
Expression constructs that comprise an isolated nucleic acid encoding a protein having an amino acid sequence of SEQ ID NO: 2 or the mature protein portion thereof and a promoter operably linked to the polynucleotide also form part of the invention. In specific embodiments, the expression construct is such that the nucleic acid comprises a mature protein coding sequence of SEQ ID NO: 1. The expression construct is an expression construct selected from the group consisting of an adenoassociated viral construct, an adenoviral construct, a herpes viral expression construct, a vaccinia viral expression construct, a retroviral expression construct, a lentiviral expression construct and a naked DNA expression construct.
Also part of the invention are recombinant host cells stably transformed or transfected with a nucleic acid or an expression construct of the invention in a manner that allows the expression in the host cell of a protein of SEQ ID NO: 2. Preferably, the nucleic acid transforming the host cell comprises a mature protein encoding sequence of SEQ ID NO: 1. Recombinant host cells stably transformed or transfected with an expression construct of the invention in a manner allowing the expression in the host cell of a protein product of the expression construct also are contemplated.
The host cells may be mammalian, a bacterial, yeast cells, or insect cells. It may be advantageous that the recombinant host cells produced by the invention further express one or more β-subunits of a sodium channel selected from the group consisting of β1, β2, β3 and β4. In specific embodiments, the host cell is a HEK293 cell line.
The invention further provides an isolated and purified protein comprising an amino acid sequence of an amino acid sequence of SEQ ID NO: 2. In particular embodiments, the isolated and purified protein comprises an amino acid sequence that is 99% identical to the complete sequence of SEQ ID NO: 2. In other embodiments, the isolated and purified protein comprises an amino acid sequence that is 95% identical to the complete sequence of SEQ ID NO: 2.
The invention also comprises a diagnostic kit for detecting a nucleic acid that encodes a sodium channel α-subunit polypeptide, the polypeptide being encoded by the sequence of SEQ ID NO: 1 comprising an isolated nucleic acid probe complementary to the complete sequence of SEQ ID NO: 1, and means for containing the nucleic acid.
Methods of identifying a modulator of a chimp sodium channel α-subunit expression or activity are contemplated wherein the modulator is identified by a method comprising the steps of contacting a cell that expresses a nucleic acid of SEQ ID NO: 1 with the candidate modulator substance; monitoring the expression or ion channel activity of a protein of SEQ ID NO: 2; and comparing the expression or ion channel activity of a protein of SEQ ID NO: 2 in the presence and absence of the candidate substance; wherein an alteration in the expression or ion channel activity of a protein of SEQ ID NO: 2 indicates that the substance is a modulator of chimp sodium channel α-subunit expression or activity.
The modulator of chimp sodium channel α-subunit expression or activity may be a small molecule ion channel blocker or inhibitor, an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of the RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, a chimeric oligonucleotide or an expression construct that produces a modulatory nucleic acid (e.g., siRNA) or polypeptide.
The invention also provides methods of identifying a test compound that binds to a sodium channel comprising providing a cell that expresses a sodium channel having a sequence of SEQ ID NO: 2; contacting the host cell with the test compound and determining the binding of the test compound to the sodium channel; and comparing the binding of the test compound to the host cell determined in step (b) to the binding of the test compound with a cell that does not express a sodium channel.
Also provided is an assay for identifying a test compound that modulates the activity of a sodium channel comprising providing a host cell that expresses a functional sodium channel comprising at least one polypeptide comprising the amino acid sequence of SEQ ID NO: 2; contacting the host cell with a test compound under conditions that would activate sodium channel activity of the functional sodium channel in the absence of the test compound; and determining whether the host cell contacted with the test compound exhibits a modulation in activity of the functional sodium channel. In particular embodiments, the host cell has been genetically engineered to express or overexpress the functional sodium channel. In other embodiments, the host cell has been genetically engineered by the introduction into the cell of a nucleic acid molecule having a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. Preferably, the host cell has been genetically engineered to upregulate the expression of a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In particular embodiments, the upregulated nucleic acid is endogenous to the host cell. Preferably, the modulation of the functional sodium channel activity is an inhibition of that activity.
A method of producing a purified chimp sodium channel α-subunit protein also is provided, the method comprising preparing an expression construct comprising a nucleic acid of SEQ ID NO: 1 operably linked to a promoter; transforming or transfecting a host cell with the expression construct in a manner effective to allow the expression of a protein having an amino acid sequence of SEQ ID NO: 2, or the mature protein portion thereof in the host cell; culturing the transformed or transfected cell under conditions to allow the production of the protein by the transformed or transfected host cell; and isolating the chimp sodium channel α-subunit protein from the host cell.
Other embodiments contemplate treatment of a disorder by administering to a subject in need thereof a pharmaceutical composition that comprises a compound identified according to the methods described herein and a pharmaceutically acceptable carrier, excipient or diluent.
Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.